Structures of Saccharolobus solfataricus initiation complexes with leaderless mRNAs highlight archaeal features and eukaryotic proximity.

The archaeal ribosome is of the eukaryotic type. TACK and Asgard superphyla, the closest relatives of eukaryotes, have ribosomes containing eukaryotic ribosomal proteins not found in other archaea, eS25, eS26 and eS30. Here, we investigate the case of Saccharolobus solfataricus, a TACK crenarchaeon, using mainly leaderless mRNAs.

Visualization of translation reorganization upon persistent ribosome collision stress in mammalian cells.

Aberrantly slow ribosomes incur collisions, a sentinel of stress that triggers quality control, signaling, and translation attenuation. Although each collision response has been studied in isolation, the net consequences of their collective actions in reshaping translation in cells is poorly understood. Here, we apply cryoelectron tomography to visualize the translation machinery in mammalian cells during persistent collision stress.

Redesigning methionyl-tRNA synthetase for β-methionine activity with adaptive landscape flattening and experiments.

Amino acids (AAs) with a noncanonical backbone would be a valuable tool for protein engineering, enabling new structural motifs and building blocks. To incorporate them into an expanded genetic code, the first, key step is to obtain an appropriate aminoacyl-tRNA synthetase. Currently, directed evolution is not available to optimize AAs with noncanonical backbones, since an appropriate selective pressure has not been discovered.

Binding of human Cdc123 to eIF2γ.

Eukaryotic initiation factor 2 (eIF2) plays a key role in protein synthesis and in its regulation. The assembly of this heterotrimeric factor is facilitated by Cdc123, a member of the ATP grasp family that binds the γ subunit of eIF2. Notably, some mutations related to MEHMO syndrome, an X-linked intellectual disability, affect Cdc123-mediated eIF2 assembly.

Visualization of translation reorganization upon persistent collision stress in mammalian cells.

Aberrantly slow mRNA translation leads to ribosome stalling and subsequent collision with the trailing neighbor. Ribosome collisions have recently been shown to act as stress sensors in the cell, with the ability to trigger stress responses balancing survival and apoptotic cell-fate decisions depending on the stress level. However, we lack a molecular understanding of the reorganization of translation processes over time in mammalian cells exposed to an unresolved collision stress.

Structural insights into the evolution of late steps of translation initiation in the three domains of life.

In eukaryotes and in archaea late steps of translation initiation involve the two initiation factors e/aIF5B and e/aIF1A. These two factors are also orthologous to the bacterial IF2 and IF1 proteins, respectively. Recent cryo-EM studies showed how e/aIF5B and e/aIF1A cooperate on the small ribosomal subunit to favor the binding of the large ribosomal subunit and the formation of a ribosome competent for elongation.

Grid batch-dependent tuning of glow discharge parameters.

Sample preparation on cryo-EM grids can give various results, from very thin ice and homogeneous particle distribution (ideal case) to unwanted behavior such as particles around the "holes" or complexes that do not entirely correspond to the one in solution (real life). We recently run into such a case and finally found out that variations in the 3D reconstructions were systematically correlated with the grid batches that were used. We report the use of several techniques to investigate the grids' characteristics, namely TEM, SEM, Auger spectroscopy and Infrared Interferometry.

Medical contrast agents as promising tools for biomacromolecular SAXS experiments.

Small-angle X-ray scattering (SAXS) has become an indispensable tool in structural biology, complementing atomic-resolution techniques. It is sensitive to the electron-density difference between solubilized biomacromolecules and the buffer, and provides information on molecular masses, particle dimensions and interactions, low-resolution conformations and pair distance-distribution functions. When SAXS data are recorded at multiple contrasts, i.

Role of aIF5B in archaeal translation initiation.

In eukaryotes and in archaea late steps of translation initiation involve the two initiation factors e/aIF5B and e/aIF1A. In eukaryotes, the role of eIF5B in ribosomal subunit joining is established and structural data showing eIF5B bound to the full ribosome were obtained. To achieve its function, eIF5B collaborates with eIF1A.

A millisecond passive micromixer with low flow rate, low sample consumption and easy fabrication.

Fast mixing of small volumes of solutions in microfluidic devices is essential for an accurate control and observation of the dynamics of a reaction in biological or chemical studies. It is often, however, a challenging task, as the Reynolds number (Re) in microscopic devices is typically < 100. In this report, we detail a novel mixer based on the "staggered herring bone" (SHB) pattern and "split-recombination" strategies with an optimized geometry, the periodic rotation of the flow structure can be controlled and recombined in a way that the vortices and phase shifts of the flow induce intertwined lamellar structures, thus increasing the contact surface and enhancing mixing.

A novel minimal motif for left-handed G-quadruplex formation.

A recent study on the left-handed G-quadruplex (LHG4) DNA revealed a 12-nt minimal motif GTGGTGGTGGTG with the ability to independently form an LHG4 and to drive an adjacent sequence to LHG4 formation. Here we have identified a second LHG4-forming motif, GGTGGTGGTGTG, and determined the X-ray crystal structure of an LHG4 involving this motif. Our structural analysis indicated the role of split guanines and single thymine loops in promoting LHG4 formation.

Bulges in left-handed G-quadruplexes.

G-quadruplex (G4) DNA structures with a left-handed backbone progression have unique and conserved structural features. Studies on sequence dependency of the structures revealed the prerequisites and some minimal motifs required for left-handed G4 formation. To extend the boundaries, we explore the adaptability of left-handed G4s towards the existence of bulges.

Recent Advances in Archaeal Translation Initiation.

Translation initiation (TI) allows accurate selection of the initiation codon on a messenger RNA (mRNA) and defines the reading frame. In all domains of life, translation initiation generally occurs within a macromolecular complex made up of the small ribosomal subunit, the mRNA, a specialized methionylated initiator tRNA, and translation initiation factors (IFs). Once the start codon is selected at the P site of the ribosome and the large subunit is associated, the IFs are released and a ribosome competent for elongation is formed.

SpiCee: A Genetic Tool for Subcellular and Cell-Specific Calcium Manipulation.

Calcium is a second messenger crucial to a myriad of cellular processes ranging from regulation of metabolism and cell survival to vesicle release and motility. Current strategies to directly manipulate endogenous calcium signals lack cellular and subcellular specificity. We introduce SpiCee, a versatile and genetically encoded chelator combining low- and high-affinity sites for calcium.

Structural basis of the interaction between cyclodipeptide synthases and aminoacylated tRNA substrates.

Cyclodipeptide synthases (CDPSs) catalyze the synthesis of various cyclodipeptides by using two aminoacyl-tRNA (aa-tRNA) substrates in a sequential mechanism. Here, we studied binding of phenylalanyl-tRNA to the CDPS from (-CDPS) by gel filtration and electrophoretic mobility shift assay. We determined the crystal structure of the -CDPS:Phe-tRNA complex to 5 Å resolution and further studied it in solution using small-angle X-ray scattering (SAXS).

Cryo-EM study of an archaeal 30S initiation complex gives insights into evolution of translation initiation.

Archaeal translation initiation occurs within a macromolecular complex containing the small ribosomal subunit (30S) bound to mRNA, initiation factors aIF1, aIF1A and the ternary complex aIF2:GDPNP:Met-tRNA. Here, we determine the cryo-EM structure of a 30S:mRNA:aIF1A:aIF2:GTP:Met-tRNA complex from Pyrococcus abyssi at 3.2 Å resolution.

Adaptive landscape flattening allows the design of both enzyme: Substrate binding and catalytic power.

Designed enzymes are of fundamental and technological interest. Experimental directed evolution still has significant limitations, and computational approaches are a complementary route. A designed enzyme should satisfy multiple criteria: stability, substrate binding, transition state binding.

Use of β-methionine as an amino acid substrate of Escherichia coli methionyl-tRNA synthetase.

Polypeptides containing β-amino acids are attractive tools for the design of novel proteins having unique properties of medical or industrial interest. Incorporation of β-amino acids in vivo requires the development of efficient aminoacyl-tRNA synthetases specific of these non-canonical amino acids. Here, we have performed a detailed structural and biochemical study of the recognition and use of β-Met by Escherichia coli methionyl-tRNA synthetase (MetRS).

Recognition of different base tetrads by RHAU (DHX36): X-ray crystal structure of the G4 recognition motif bound to the 3'-end tetrad of a DNA G-quadruplex.

G-quadruplexes (G4) are secondary structures of nucleic acids that can form in cells and have diverse biological functions. Several biologically important proteins interact with G-quadruplexes, of which RHAU (or DHX36) - a helicase from the DEAH-box superfamily, was shown to bind and unwind G-quadruplexes efficiently. We report a X-ray co-crystal structure at 1.

SponGee: A Genetic Tool for Subcellular and Cell-Specific cGMP Manipulation.

cGMP is critical to a variety of cellular processes, but the available tools to interfere with endogenous cGMP lack cellular and subcellular specificity. We introduce SponGee, a genetically encoded chelator of this cyclic nucleotide that enables in vitro and in vivo manipulations in single cells and in biochemically defined subcellular compartments. SponGee buffers physiological changes in cGMP concentration in various model systems while not affecting cAMP signals.

NMR solution and X-ray crystal structures of a DNA molecule containing both right- and left-handed parallel-stranded G-quadruplexes.

Analogous to the B- and Z-DNA structures in double-helix DNA, there exist both right- and left-handed quadruple-helix (G-quadruplex) DNA. Numerous conformations of right-handed and a few left-handed G-quadruplexes were previously observed, yet they were always identified separately. Here, we present the NMR solution and X-ray crystal structures of a right- and left-handed hybrid G-quadruplex.

Start Codon Recognition in Eukaryotic and Archaeal Translation Initiation: A Common Structural Core.

Understanding molecular mechanisms of ribosomal translation sheds light on the emergence and evolution of protein synthesis in the three domains of life. Universally, ribosomal translation is described in three steps: initiation, elongation and termination. During initiation, a macromolecular complex assembled around the small ribosomal subunit selects the start codon on the mRNA and defines the open reading frame.

A Minimal Sequence for Left-Handed G-Quadruplex Formation.

Recently, we observed the first example of a left-handed G-quadruplex structure formed by natural DNA, named Z-G4. We analysed the Z-G4 structure and inspected its primary 28-nt sequence in order to identify motifs that convey the unique left-handed twist. Using circular dichroism spectroscopy, NMR spectroscopy, and X-ray crystallography, we revealed a minimal sequence motif of 12 nt (GTGGTGGTGGTG) for formation of the left-handed DNA G-quadruplex, which is found to be highly abundant in the human genome.

Role of aIF1 in Pyrococcus abyssi translation initiation.

In archaeal translation initiation, a preinitiation complex (PIC) made up of aIF1, aIF1A, the ternary complex (TC, e/aIF2-GTP-Met-tRNAiMet) and mRNA bound to the small ribosomal subunit is responsible for start codon selection. Many archaeal mRNAs contain a Shine-Dalgarno (SD) sequence allowing the PIC to be prepositioned in the vicinity of the start codon. Nevertheless, cryo-EM studies have suggested local scanning to definitely establish base pairing of the start codon with the tRNA anticodon.